Hyaluronan (HA, hyaluronic acid) is a linear unbranched polymer made up of repeating disaccharide units of D-glucuronic acid (xcex21xe2x86x923) N-acetylglucosamine (xcex21xe2x86x924). HA biosynthesis requires two enzyme activities; the transfer of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcUA), respectively, to the growing HA chain. HA is synthesized at the inner face of the plasma membrane and is subsequently extruded to the outside of the cell. HA is a major constituent of the extracellular matrix during embryonic development. For example, within the developing embryo, HA accumulates at sites of cell migration and proliferation, and has been proposed to play important roles in craniofacial, limb, neural tube, and heart development. In particular, HA is essential for the formation of endocardial cushions, the structures required for septation and the development of heart valves. In adults, HA is a major constituent of the extracellular matrix of most tissues and organs, and a critical component of the vitreous humor of the eye, joint fluid and cartilage.
HA is highly biocompatible and completely biodegradable, and has demonstrated beneficial effects when administered to the joints of arthritic race horses and to perforated rat tympanic membranes. HA has also been employed to protect eye tissue during artificial intraocular lens implantations, as a delivery agent for drugs and to prevent post-operative scarring.
Genes which encode HA biosynthetic enzymes have been identified in bacteria, e.g., Group A Streptococcus (Wessels et al., Infect. Immun., 62, 433 (1994); DeAngelis et al., J. Biol. Chem., 268, 19181 (1993); DeAngelis et al., Biochemistry, 33, 9033 (1994)). Polymerization of HA by S. pyogenes occurs through the action of a single enzyme, HA synthase, encoded by the hasA gene. The S. pyogenes HA synthase is localized to the membrane and is predicted to have several transmembrane domains and a large intracellular loop encompassing the active site of the enzyme. Purified immobilized HasA has been shown to be sufficient for HA polymerization in vitro (DeAngelis et al., Biochemistry, 33, 9033 (1994)). The transfer of the hasA gene and a second gene, hasB, into heterologous bacterial species results in the synthesis of an HA capsule (DeAngelis et al., J. Biol., Chem., 268, 19181 (1993)). The hasB gene encodes a UDP-glucose dehydrogenase, which converts UDP-glucose to UDP-glucuronic acid (UDP-GlcUA), a subunit of HA.
However, there is evidence that other genes are also involved in bacterial HA biosynthesis. A protein originally identified in Streptococcus equisimilis as HA synthase (Lansing et al., Biochem. J., 289, 179 (1993)) has no sequence similarity to S. pyogenes HasA but has significant sequence similarity to bacterial proteins involved in oligopeptide binding and transport. Although the total amount of HA synthesized by bacterial cells overexpressing the S. equisimilis HA synthase increased, the length of the resultant HA chains was significantly shorter, suggesting that the increase may be a function of an elevation in the rate of HA transport from the cell (O""Regan et al., Int. J. Biol. Macromol., 16, 283 (1994)). Thus, rather than being directly involved in HA biosynthesis, the S. equisimilis HA synthase may be involved in the transport of HA, or may participate in HA synthesis as an accessory molecule, rather than as the synthase itself.
While both bacterial and animal sources of HA exist, high molecular weight HA is difficult and costly to isolate and purify due to the fact that HA is complexed with proteoglycans. Moreover, both bacterial and animal sources of HA are increasingly under more stringent regulatory controls due to fear of contamination with identifiable, or as yet unidentified, infectious or toxic agents. Furthermore, the extensive purification process of HA polymer from cells results in an HA polymer of considerable molecular weight polydispersity
Thus, there is a need to isolate and purify genes that encode eukaryotic HA biosynthetic enzymes or proteins associated with the extracellular accumulation of HA.
The present invention provides an isolated and purified DNA molecule comprising a preselected DNA segment encoding eukaryotic, preferably mammalian, hyaluronan synthase-2 (Has2), or which encodes a biologically active subunit thereof. A preferred embodiment of the invention is a DNA molecule comprising a preselected DNA segment, e.g., SEQ ID NO:1, that encodes murine hyaluronan synthase-2. A murine hyaluronan synthase-2 having SEQ ID NO:2 has 21% identity and 28% similarity to Streptococcal HasA, and 55% identity and 73% similarity to murine Has1 (Itano et al., J. Biol. Chem., 271, 9875 (1996)). Because the deduced amino acid sequence of Has1 is distinct from the murine hyaluronan synthase-2 having SEQ ID NO:2, there appears to be more than one mammalian gene encoding an enzyme or protein which is associated with HA biosynthesis and/or extracellular HA accumulation. Another preferred embodiment of the invention is a DNA molecule comprising a preselected DNA segment, e.g., SEQ ID NO:23, that encodes human hyaluronan synthase-2. Also provided is an isolated and purified DNA molecule comprising a preselected DNA segment which encodes a protein that increases the amount of extracellular hyaluronan produced by cultured primate cells transformed so as to express said DNA segment.
Further provided is an isolated and purified DNA molecule comprising a DNA segment encoding eukaryotic, preferably mammalian, hyaluronan synthase-3 (Has3), or a biologically active subunit thereof. A preferred embodiment of the invention includes a preselected DNA segment comprising SEQ ID NO:31 which encodes a murine hyaluronan synthase-3 comprising SEQ ID NO:32. Another preferred embodiment of the invention includes a DNA molecule comprising a preselected DNA segment comprising SEQ ID NO:25 which encodes a human hyaluronan synthase-3 comprising SEQ ID NO:29. The DNA molecules of the invention are double-stranded or single-stranded, preferably, they are cDNA.
An isolated and purified DNA molecule, such as a probe or a primer, of at least seven, preferably at least fifteen, nucleotide bases which hybridizes under stringent conditions to the DNA molecules of the invention, or RNA molecules derived from these DNA molecules, is also provided by the invention. The term xe2x80x9cstringent conditionsxe2x80x9d is defined hereinbelow. The probes or primers of the invention have at least about 80%, preferably at least about 90%, identity to the above-disclosed DNA sequences. A preferred embodiment of the invention includes a probe or primer which has at least about 80%, preferably at least about 90%, identity to 1) SEQ ID NO:25, 2) SEQ ID NO:26 or 3) SEQ ID NO:31, or a sequence complementary thereto. The probes or primers of the invention are detectably labeled or have a binding site for a detectable label. Such probes or primers are useful to detect, quantify and/or amplify DNA strands with complementary to sequences related to hyaluronan synthase-2 or hyaluronan synthase-3 in eukaryotic tissue samples.
The present invention also provides an expression cassette comprising a promoter which is functional in a host cell operably linked to a preselected DNA segment encoding hyaluronan synthase-2. Preferably, the expression cassette comprises a preselected DNA segment encoding murine hyaluronan synthase-2. Another preferred embodiment of the invention is an expression cassette comprising a preselected DNA segment encoding human hyaluronan synthase-2.
The present invention further provides an expression cassette comprising a promoter which is functional in a host cell operably linked to a preselected DNA segment encoding hyaluronan synthase-3. Preferably, the expression cassette comprises a preselected DNA segment encoding murine hyaluronan synthase-3. Another preferred embodiment of the invention is an expression cassette comprising a preselected DNA segment encoding human hyaluronan synthase-3. Such expression cassettes can be placed into expression vectors which can then be employed to transform prokaryotic or eukaryotic host cells. The present vectors can also contain a functional DNA sequence which is a selectable marker gene or reporter gene, as described below.
Also provided is a transformed host cell, the genome of which has been augmented by a preselected DNA sequence encoding hyaluronan synthase-2, a preselected DNA sequence encoding hyaluronan synthase-3, or a combination thereof. Preferably, the preselected DNA sequence is integrated into the chromosome of the transformed host cell, and is heritable.
Expression of mouse hyaluronan synthase-2 or mouse hyaluronan synthase-3 in COS-1 cultured primate cells results in the formation of large well-pronounced HA coats, as described hereinbelow. Moreover, HA coat formation in COS cells transfected with an hyaluronan synthase-2 expression vector occurred in the absence of HA receptor expression, exogenously added HA, or proteoglycans. This suggests that hyaluronan synthase-2 expression leads to the synthesis of HA, in a form which is extruded through the plasma membrane and may associate with the cell surface to form an HA coat through continued attachment to the HA synthase.
Further provided is isolated, purified hyaluronan synthase-2 polypeptide. A preferred embodiment of the invention is isolated, purified murine hyaluronan synthase-2 polypeptide. A more preferred embodiment of the invention is isolated, purified murine hyaluronan synthase-2 polypeptide having SEQ ID NO:2.
Also provided is isolated, purified hyaluronan synthase-3 polypeptide. A preferred embodiment of the invention is isolated, purified murine hyaluronan synthase-3 polypeptide. A more preferred embodiment of the invention is isolated, purified murine hyaluronan synthase-3 polypeptide having SEQ ID NO:32.
As used herein, the term xe2x80x9cHas2xe2x80x9d or xe2x80x9chyaluronan synthase-2xe2x80x9d is preferably defined to mean a polypeptide comprising SEQ ID NO:2, as well as variants of SEQ ID NO:2 which have at least about 80%, preferably at least about 90%, identity or homology to SEQ ID NO:2, or a biologically active subunit thereof. Biologically active subunits of hyaluronan synthase-2, variant hyaluronan synthase-2 polypeptides and biologically active subunits thereof, falling within the scope of the invention have at least about 50%, preferably at least about 80%, and more preferably at least about 90%, the activity of the hyaluronan synthase-2 polypeptide comprising SEQ ID NO:2. The activity of an hyaluronan synthase-2 polypeptide can be measured by methods well known to the art including, but not limited to, the particle exclusion assay described hereinbelow, an immunoassay which detects HA production, as described by Itano et al. (J. Biol. Chem., 271, 9875 (1996)), HA synthase activity of crude membrane preparations, as described by Itano et al. (supra), or HA synthase activity of cell lysate preparations, as described by Meyer et al. (Proc. Natl. Acad. Sci. USA, 93, 4543 (1996)).
As used herein, the term xe2x80x9cHas3xe2x80x9d or xe2x80x9chyaluronan synthase-3xe2x80x9d is preferably defined to mean a polypeptide comprising SEQ ID NO:32, SEQ ID NO:29, or a biologically active subunit thereof, as well as variants of SEQ ID NO:32 or SEQ ID NO:29 and subunits thereof which have at least about 80%, preferably at least about 90%, identity or homology to SEQ ID NO:32 or SEQ ID NO:29, respectively. Biologically active subunits of hyaluronan synthase-3, variant hyaluronan synthase-3 polypeptides and biologically active subunits thereof, falling within the scope of the invention have at least about 50%, preferably at least about 80%, and more preferably at least about 90%, the activity of the hyaluronan synthase-3 polypeptide comprising SEQ ID NO:32 or SEQ ID NO:29. The activity of an hyaluronan synthase-3 polypeptide can be measured by the methods described above for hyaluronan synthase-2.
The present invention also provides a method to produce hyaluronan synthase-2, comprising: culturing a host cell transformed with a nucleic acid molecule comprising a DNA segment encoding hyaluronan synthase-2 operably linked to a promoter, so that said host cell expresses said hyaluronan synthase-2. The method also preferably provides isolated recombinant hyaluronan synthase-2 polypeptide which is recovered from the transformed host cells.
Also provided is a method to produce hyaluronan synthase-3, comprising: culturing a host cell transformed with a nucleic acid molecule comprising a DNA segment encoding hyaluronan synthase-3 operably linked to a promoter, so that said host cell expresses said hyaluronan synthase-3. The method also preferably provides isolated recombinant hyaluronan synthase-3 polypeptide which is recovered from the transformed host cells. Optionally, host cells can be co-transformed with a nucleic acid molecule comprising a DNA segment encoding hyaluronan synthase-3 operably linked to a promoter and a nucleic acid molecule comprising a DNA segment encoding hyaluronan synthase-2 operably linked to a promoter.
Further provided is a method of altering the amount of hyaluronan produced by a cell. The method comprises introducing into a host cell a preselected DNA segment encoding hyaluronan synthase-2 operably linked to a promoter so as to yield a transformed host cell. The preselected DNA segment is expressed as hyaluronan synthase-2 in the transformed host cell in an amount that results in the transformed host cell producing an altered, preferably increased, amount of hyaluronan relative to the amount of hyaluronan produced by a corresponding untransformed host cell.
Also provided is a method of altering the amount of hyaluronan produced by a cell. The method comprises introducing into a host cell a preselected DNA segment encoding hyaluronan synthase-3 operably linked to a promoter so as to yield a transformed host cell. The preselected DNA segment is expressed as hyaluronan synthase-3 in the transformed host cell in an amount that results in the transformed host cell producing an altered, preferably increased, amount of hyaluronan relative to the amount of hyaluronan produced by a corresponding untransformed host cell.
Once isolated and purified, the genes involved in HA biosynthesis and extracellular accumulation of HA can be employed to synthesize HA in vitro. Because in vitro synthesized HA is of extremely high purity, is free from bacterial and animal cell contaminants, and can be optimized as to its physicochemical properties, it is a preferred source of HA relative to HA derived from bacterial or animal sources.
Moreover, the identification of genes involved in HA biosynthesis and/or coat formation may also be useful for defining the molecular basis for genetic diseases which are associated with a deficiency in HA biosynthesis, such as cartilage pathologies, for providing a clinically useful diagnostic test or in molecular-based therapeutics. Furthermore, the cloning of these genes will help to elucidate the molecular mechanism giving rise to the alteration of the protein encoded by the gene in patients having a particular disorder, e.g., a cartilage deficiency associated with reduced HA biosynthesis.
The probes and primers of the present invention are useful for detecting the expression of the DNA molecules of the present invention, detecting related DNA molecules and amplifying nucleic acid sequences that fall within the scope of the present invention.
The present invention also provides isolated and purified DNA molecules which provide xe2x80x9canti-sensexe2x80x9d mRNA transcripts of the DNA sequences, including SEQ ID NO:1 or SEQ ID NO:31, which, when expressed from an expression cassette in a host cell, can alter HA expression.